sodium;2-hexadecylsulfanyl-1H-benzimidazole-5-sulfonate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

yes

Remarks:

Cell densities were determined using a spectrophotometer with immersion probe (path length = 10 mm instead of 20 mm). Evaluation: A new spectrophotometer was used. This has no impact on the test interpretation

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

sodium;2-hexadecylsulfanyl-1H-benzimidazole-5-sulfonate

EC Number:

616-081-6

Cas Number:

743423-33-8

Molecular formula:

C23 H37 N2 Na O3 S2

IUPAC Name:

sodium;2-hexadecylsulfanyl-1H-benzimidazole-5-sulfonate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 2.0 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Remarks:

M2; according to the OECD 201 Guideline, formulated using Milli-RO water

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

7.9-8.2

Nominal and measured concentrations:

WAFs individually prepared at loading rates of 1.0, 10 and 100 mg/L.
Samples taken from the control and the highest test concentration were analysed. At the start of the test, the actual test concentration was 0.52 mg/L in the WAF prepared at 100 mg/L. At the end of the exposure period, the measured concentration was at 80% of the initial concentration (0.41 mg/L). The concentrations measured in the samples without algae were comparable.
It should be noted that small responses were measured in the samples taken from the control, which likely derived from contaminations during the sample preparation and analysis. Based on these results, the measured concentration was used to determine the effect parameters.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Type (delete if not applicable): Capped vessels
- Aeration: no
- Initial cells density: An initial cell density of 1 x 10^4 cells/mL.
- Control end cells density:
- No. of organisms per vessel: 1 x 10^4 cells/mL.
- No. of vessels per concentration (replicates): 6 for the highest concentration, 3 for the intermediate concentrations
- No. of vessels per control (replicates): 6
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.


GROWTH MEDIUM
- Standard medium used: yes: M2


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 87 to 89 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm).
- Chlorophyll measurement: no
- Other: At the end of the test, microscopic observations were performed on the control and the highest test concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations:
0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: the combined limit/range-finding study was sufficient to determine effect levels.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Details on results:

No inhibition of algal growth was observed at the end of the test.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.52 mg/L when compared to the control.

Samples taken from the control and the highest test concentration were analysed. At the start of the test, the actual test concentration was 0.52 mg/L in the WAF prepared at 100 mg/L. At the end of the exposure period, the measured concentration was at 80% of the initial concentration (0.41 mg/L). The concentrations measured in the samples without algae were comparable.
It should be noted that small responses were measured in the samples taken from the control, which likely derived from contaminations during the sample preparation and analysis.
Based on these results, the measured concentration was used to determine the effect parameters.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.34 mg/L with a 95% confidence interval ranging from 0.34 to 0.35 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Any other information on results incl. tables

Effect Parameters

Parameter (mg/L)	NOEC	EC10	EC50
Growth rate	0.52	>0.52	>0.52
Yield	0.52	>0.52	>0.52

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of V182675 tested.
The EC50 for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. exceeded a measured concentration of 0.52 mg/L corresponding to the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.
The EC50 for yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a measured concentration of 0.52 mg/L corresponding to the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.
The 72h-NOEC for both growth rate and yield inhibition was 0.52 mg/L.

Executive summary:

The objectiveofthe study was to evaluate V182675 for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀and EC₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of V182675 tested was an off-white powder with lumps and a purity of 92.7% and not completely soluble in test medium at the loading rates initially prepared.

Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0 to 100 mg/L and used as test concentrations.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control andto a WAF prepared at a loading rate of 100 mg/Lin the limit test. In addition, three replicates per group were exposed to WAFs individually prepared at 1.0 and 10mg V182675 per litre in a combined range-finding test. The initial algal cell density was 10⁴ cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from the control and the highest test concentration were analysed. At the start of the test, the actual test concentration was 0.52 mg/L in the WAF prepared at 100 mg/L. At the end of the exposure period, the measured concentration was 80% of the initial concentration (0.41 mg/L). Based on these results, the measured concentrations were used to determine the effect parameters.

No inhibition of algal growth was observed at the end of the test.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)	NOEC	EC10	EC50
Growth rate	0.52	>0.52	>0.52
Yield	0.52	>0.52	>0.52

 

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of V182675 tested.